Plants are under constant attack from a range of pathogens, yet disease symptoms are comparatively rare. Cell-autonomous innate immunity ensures each plant cell has the ability to respond to pathogen attack. In contrast, animals use a circulatory system to ensure full spatial coverage of innate immunity, while jawed vertebrates have supplemented this defense through the evolution of an adaptive immune system. Despite the presence of similar innate immunity strategies in plants and animals, the precise epitopes perceived differ. Thus, instead of divergent evolution from a common ancestor, any similarities are likely to be a result of convergent evolution (Zipfel and Felix, G. (2005) Current Opinion in Plant Biology 8:353-360; Ausubel (2005) Nature Immunology 6:973-979). In plants, innate immunity consists of three main defense systems: physical, local and systemic. Physical defense include the waxy cuticle and rigid cell wall, as well as secondary metabolites and enzymes possessing anti-microbial properties. This defense is partially breached by stomata or through wounding. Recognition at the local level then relies on the specific perception of microbial compounds. Pattern-recognition receptors (PRRs) recognise pathogen-associated molecular patterns (PAMPs), resulting in PAMP-triggered immunity (PTI). Virulent pathogens secrete effectors into the plant cytosol to suppress PTI. However resistance (R) proteins perceive these microbial effectors to prompt effector-triggered immunity (ETI). These defenses may be breached by further pathogen effectors designed to suppress ETI. The ‘zig-zag’ model summarises these molecular interactions as five phases occurring over the dynamic coevolution between the plant and its pathogens (Jones and Dangl (2006) Nature 444:323-328; Chisholm et al. (2006) Cell 124: 803-814). In addition, systemic defense responses induced by PAMPs and/or effector proteins prepare naïve tissues for further attack. Together these three facets form an effective defensive network responsible for the phenomenon whereby most plants are resistant to a majority of microbes (Nürnberger et al. (2004) Immunological Reviews 198:249-266).
The primary immune response to bacterial invasion that results in PTI begins with the recognition of PAMPs by PPRs. PTI comprises numerous well-characterized responses including increases in calcium, ethylene, reactive oxygen species (ROS) and extracellular pH, activation of mitogen-activated protein kinase (MAPK) cascades and defense gene expression, deposition of callose, and the inhibition of seedling growth (Ma and Berkowitz (2007) Cellular Microbiology 9:2571-2585; Chisholm et al. (2006) Cell 124:803-814; Nürnberger et al. (2004) Immunological Reviews 198:249-266). These responses are common to elicitation by many bacterial PAMPs, as well as those from fungi and oomycetes. Thus PAMPs are conserved microbial feature which indicate generic danger; it is unlikely the signal conveys information that would allow the plant to distinguish between pathogens (Zipfel et al. (2006) Cell 125:749-760).
Over recent years, many potential plant PAMPs have been discovered, whereas the corresponding PRRs have been harder to identify. It has become clear that the breadth of perception of individual PAMPs differs dramatically. Out of the bacterial PAMPs, flagellin is recognised by most plant species whereas responses to elongation factor-Tu (EF-Tu) and the cold-shock protein CSP are restricted to the families Brassicaceae and Solanaceae respectively (Zipfel and Felix, G. (2005) Current Opinion in Plant Biology 8:353-360). This difference in perception is indicative of divergent evolution between plant families. An evolutionary arms race occurs whereby the bacterial PAMP evolves to avoid recognition while the plant PRR counter-evolves to increase sensitivity. However plants can perceive multiple PAMPs per microbe. The plant model Arabidopsis thaliana (At), for example, can perceive the bacterial PAMPs flagellin, EF-Tu, lipopolysacharides (LPS) and peptidoglycans (PGN), as well as the fungal PAMPs chitin octamers and ethylene-inducing xylanase (EIX). However the Arabidopsis thaliana receptors responsible for this recognition have so far only been identified for flagellin and EF-Tu. In tomato, the receptor LeEIX1/2 perceives chitin, while the rice receptor CEBiP recognises EIX (Kaku et al. (2006) Proc Natl Acad Sci USA 103:11086-1109; Ron and Avni (2004) Plant Cell 16:1604-1615). But although clear homologues exist in At, their role is not proven. However the receptor-like kinase, CERK1 (also known as LysM RLK1) is essential for chitin elicitor signaling in Arabidopsis thaliana (Miya et al. (2007) Proc Natl Acad Sci USA 104:19613-19618; Wan et al. (2008) Plant Cell 20:471-381). Meanwhile plant receptors for LPS and PGN are still entirely unknown. (Newman et al. (2007) Journal of Endotoxin Research 13:69-84; Gust et al. (2007) J Biol Chem 282:32338-48).
Despite the overall effectiveness of innate immunity, plant diseases are still a major social and economic problem. There is a distinct need for effective and stable disease control methods, particularly for Proteobacteria which include the Pseudomonas and Xanthomonas genera. Bacteria of this phylum have a broad host range in which they cause diverse symptoms affecting both crop yield and quality (Abramovitch et al. (2006) Nature Reviews Molecular Cell Biology 7:601-611). There are three main methods of human intervention. (i) Traditional approaches include: removing sources of infection; avoiding and controlling vectors by altering the time of sowing and using chemical sprays; and by breeding for increased resistance. (ii) Biotechnological approaches include producing pathogen-free seed and using biological control methods to help prevent epidemics. (iii) The third method of control is through transgenics. This powerful technique can be used to enhance plant immunity through the expression of additional genes. Transgenic plants may express plant-derived proteins such as additional receptors, signalling molecules, or antimicrobial peptides, or they may express pathogen-derived protein such as effectors. This project focuses on the potential for improving disease resistance through the transgenic expression of additional PRRs.
The PRRs FLS2 and EF-Tu receptor (EFR) are remarkable for two reasons: so far they are the only PRRs found in Arabidopsis thaliana and they are the only known PRRs for bacterial PAMPs (Zipfel et al. (2004) Nature 428: 764-767; Zipfel et al. (2006) Cell 125:749-760). Both belong to the leucine-rich repeat receptor-like kinase (LRR-RLK) family containing members which possess a common domain structure. The LRR region is known to be a key protein recognition motif in a vast variety of protein families (Kobe and Kajava (2001) Curr. Opin. Struct. Biol. 11:725-732). In PRR genes, the LRR domain is extracellular and is thought to provide the structural framework that mediates crucial protein-protein interactions. For example, the LRR in FLS2 contains residues essential for flg22 binding, enabling perception of flagellin (Chinchilla et al. (2006) The Plant Cell 18:465-476; Dunning et al. (2007) The Plant Cell 19:3297-3313). The LRR domain contains tandem copies of a LRR repeat. One LRR repeat is 20-29 residues long and contains the conserved 11-amino acid motif: LxxLxLxxN/CxL, where x can be any amino acid and L can be substituted for other hydrophobic residues in more irregular repeats (Kobe and Kajava (2001) Curr. Opin. Struct. Biol. 11:725-732). The LRR repeats are arranged so all secondary structures are parallel to a common axis, resulting in a horseshoe shape. Members of this family also contain a single-pass transmembrane domain and an intracellular Ser/Thr kinase domain which is related to Pelle in Drosophila (Shiu et al. (2001) Proc Natl Acad Sci USA 98:10763-10768). Interestingly, this is the same structure as class IV R genes such as Xa21 present in rice (Song et al. (1995) Science 270:1804-1806). This implies that R genes are adapted from more ancient PRR genes. Thus it would appear that events in the ‘zig-zag’ model occur in the order they evolved (Nürnberger et al. (2004) Immunological Reviews 198:249-266).
Recently, the EF-Tu receptor (EFR) was identified in Arabidopsis (Zipfel et al. (2006) Cell 125:749-760). EFR is a receptor kinase that is responsible for perception of EF-Tu. EFR recognizes a region of 18 amino-acids at the N-acetylated terminus of eubacterial EF-Tu that is highly conserved (Kunze et al. (2004) The Plant Cell 16:3496-3507; Zipfel et al. (2006) Cell 125:749-760). Notably eubacterial EF-Tu is different than the EF-Tu that are present in plant mitochondria and plastids, thus restricting EFR to only recognising ‘non-self’. Previous transient expression of AtEFR in Nicotiana benthamiana (Nb) showed that EFR is sufficient for EF-Tu responsiveness and that signalling components downstream of PRRs are conserved across plant families (Zipfel et al. (2006) Cell 125:749-760). However, it was unknown whether the stable expression of EPR confers recognition of EF-Tu epitope elf18 on a transformed plants and triggers activation of basal immune responses therein. It was also unknown whether this transformed plants that stably express EPR can also recognise EF-Tu present in whole pathogenic bacteria leading to enhanced disease resistance to bacterial pathogens.